RP-HPLC bubbling advice needed
 

Hi all.

I have a RP-HPLC snag. While a sample containing 30% glycerol is being run through a Synergi MaxRP-C12 column with a linear acetonitrile gradient (water phase being Tris-0.05%TFA, pH 8.5) and the gradient reaches 45-50%, pressure goes down and a bubble forms blurring a substantial part of the picture. Glycerol-free samples run with no trouble. Glycerol is essential for solubility of proteolysis fragments in the sample, sample pH is 8.0 and is needed for proteinase K proteolysis. It would be great if someone could help sort this out. Thanks in advance.

Re: RP-HPLC bubbling advice needed
 

Have you tried cleaning up your sample after lysis using a SPE cartridge or some ziptips ? Proteolysis fragments are generaly soluble in some combination of ACN/H2O and some acid (Formic or Acetic)

Re: RP-HPLC bubbling advice needed
 

Are you running your solvents through a degasser?

Re: RP-HPLC bubbling advice needed
 

Once again, thank you for your replies

Problem is I need my protein material soluble DURING proteolysis as well not just before loading it on the column. Glycerol proved to be the only reagent that kept solution clear without compromising my protease’s stability. Sucrose could be another alternative but I doubt it could prevent bubbles forming due to its high viscosity. DMSO (up to 10%) didn’t work.

We don’t have a degasser so my colleague and I were experimenting blowing helium through our buffers using available material, which didn’t help. The only protocol that worked well included a very steep linear gradient, 10 min at 1 ml/min 0-100% ACN, apparently not allowing enough time for bubbles to form but the peaks came out too stacked of course. Now that really became a problem when I started running BME-reduced samples so I finally opted for a step gradient. There’s been no bubbles with the step protocol so far (and I’m groping for wood :)) so right now I’m trying different protocols to separate both BME and urea from my reduced fragments, which is a bit tricky.

P.S. I wonder why I can’t view all of the replies to my post, only the last two. There were a lot more, were they deleted?

Re: RP-HPLC bubbling advice needed
 

did you clean it?

Re: RP-HPLC bubbling advice needed
 

First of all, I think you need to find a degasser, wherever you can get one. Do you not have access to a vacuum line? What about one of those old, highly environmentally-insensitive tap-based vacuums? Degassed solutions will make a world of difference to your purification, and will extend the life of your column.

Next, even though you may need the glycerol for the proteolysis steps, you probably won't need it during the separation. Load with glycerol, by all means, but do the gradient without any, having made sure you've removed as much as possible in a washout step.

Have you considered other columns? Buffers?

Re: RP-HPLC bubbling advice needed
 

I would expect that sparging with helium should be adequate in lieu of a degasser. So let's back up a second here... Do you visually see bubbles in the flow cell of your UV detector if you remove it and observe during the run? If it's just the pressure drop and chromatography issues there could be other explanations like a stuck check valve in one of your pumps etc.

I have never used glycerol myself, since I work entirely with small molecules, so I'm curious if it tends to act somewhat like a strong solvent in terms of elution.

Re: RP-HPLC bubbling advice needed
 

Another problem you might be having with your glycerol system is poor mixing. 30% is a heck of a lot of a fairly viscous solution, and when you mix it with acetonitrile, there's bound to be a great deal of "schlieren" mixing. Can you tell us the details of the HPLC system? Specifically, what's the size of the mixing chamber, and what flow rate are you using? How long does the gradient take?